JPH07270379A - Determination of chloride ion - Google Patents

Abstract

PURPOSE:To determine the amt. of a chloride ion by reacting a specimen containing a chloride ion with a monoamine oxidase-immobilized enzyme membrane in the presence of an amine compd. and measuring the output of an enzyme electrode generated corresponding to the reduction amt. of a reactant and the increase amt. of a reaction product. CONSTITUTION:Monoamine oxidase has a property obstructed in activity in dependence on the concn. of a chloride ion and catalyzes the production reaction of aldehyde, ammonia and hydrogen peroxide due to oxidative deamination using an amine compd. as a substrate. As a specimen, for example, blood, serum or plasma containing a chloride ion can be used. For example, an oxygen electrode is combined with an immobilized enzyme membrane prepared by immobilizing monoamine oxidase originating from Aspergillus niger on a acetylcellulose membrane to constitute an enzyme electrode. The specimen is brought into contact with the enzyme membrane in the presence of benzylamine and the output of the enzyme electrode generated to the reduction of enzyme or the increase of hydrogen peroxide accompanying the oxidation reaction of benzylamine to determine the concn. of the chloride ion.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel method for quantifying chloride ion, which can be widely used in the medical field such as clinical examination for quantifying chloride ion in body fluid.

[0002]

Background Art Chloride ions in body fluids are important ions involved in water equilibrium, acid-base equilibrium, and adjustment of osmotic pressure. Conventionally, the measurement of chlorine ions in body fluids has been performed by a colorimetric method, a coulometric titration method, an ion selective electrode method, or the like.

The colorimetric method is a method in which chlorine ions are reacted with mercuric thiocyanate and the liberated thiocyanate is colored with ferric ion for colorimetric quantification. However, there is a drawback in that

In the coulometric titration method, a constant electric current is applied to a silver electrode in an electrolytic solution to cause the liberated silver ions and chlorine ions to react with each other to precipitate as silver chloride. When all the chlorine ions are consumed, the silver ions are rapidly added. The chlorine ion is quantified from the time required for this to be taken as the end point by catching this moment with the indicator electrode, but there are drawbacks such as poor analytical processing efficiency of the sample.

The ion-selective electrode method uses an ion-selective electrode to quantify chlorine ions, but has a problem of ion specificity that it responds not only to chlorine ions but also to other halogen ions. .

Recently, a method for quantifying chloride ion by an enzymatic method using amylase has been reported [Clinical Chemistry, 34, 552, 1988]. This method does not contain harmful substances in the reagent, has good ion specificity that has been a problem in the ion selective electrode method, and has the advantage of high sample analysis efficiency if an automatic biochemical analyzer is used. Have However, since the measurement range is narrow, the stability of the reagent is poor, and the method is a colorimetric measurement method, it has a drawback that the influence of white turbidity such as bilirubin dye in serum and emulsion serum is large. In addition, the enzyme has a drawback that it cannot be used repeatedly even though it is more expensive than general chemical reagents.

[0007]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention It is difficult to be interfered by other halogen ions which have been a problem in the ion-selective electrode method, and when measuring serum, bilirubin-based dye or the like contained in a subject or cloudiness. There has been a demand for a method that can repeatedly use an enzyme without being affected by the above and can accurately quantify chloride ion by a simple operation.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that monoamine oxidase is inhibited depending on the chloride ion concentration, and further immobilization. By measuring the monoamine oxidase activity using an enzyme electrode with a monoamine oxidase membrane attached to the electrode, chlorine ion can be quantified accurately and easily,
It was confirmed that the enzyme can be used repeatedly, and the present invention has been completed.

That is, according to the present invention, an enzyme electrode comprising a combination of an immobilized enzyme membrane having a monoamine oxidase immobilized on a membrane polymer compound and an electrode is used, and the immobilized enzyme membrane of the enzyme electrode contains chlorine ions. By contacting the analyte in the presence of an amine compound, and measuring the output of the enzyme electrode corresponding to the decrease amount of the reaction product or the increase amount of the reaction product accompanying the oxidation reaction of the amine compound by the monoamine oxidase. It is a method for quantifying chlorine ions, which is characterized by determining the amount of chlorine ions.

The monoamine oxidase in the present invention is
An enzyme that has the property of being inhibited depending on the chloride ion concentration and that catalyzes the reaction of producing aldehydes, ammonia, and hydrogen peroxide by oxidative deamination using an amine compound represented by the following formula as a substrate. Amine compound + oxygen + H ₂ O → aldehyde compound +
The ammonia + hydrogen peroxide monoamine oxidase is not particularly limited as long as it is an enzyme having the above properties, and known ones can be used. Specifically, Agricultural Biological Chemistry (Agric. Biol. Chem.), 29, 649 (1965
Year) Aspergillus nige
r) -derived amine oxidase, Talaromyces flavus var. flavus (Talaromyces flavus var. flavus) described in JP-A-58-9698.
Amine oxidase derived from M4175, Petromyces alliaceus M4648
Amine oxidase derived from, Amine oxidase derived from Neosartorya fischeri M4690 (Neosartorya fischeri M4690), Eurotium thievarieri M48
Amine oxidase derived from bacterium No. 05 (Eurotium chevelieri M4805), amine oxidase derived from Eubenicillium parvum M5051 (Eupenicillium parvum M5051), and the like are mentioned. Niger (Aspergillus
niger) -derived amine oxidase can be preferably used. The target enzyme can be obtained from such a monoamine oxidase-producing bacterium by a known method.

As the membranous polymer compound used for the immobilized enzyme membrane, known acetyl cellulose, vinyl chloride resin, polycarbonate, nylon, polypropylene, polyethylene, collagen and the like can be used. Acetyl cellulose is preferred because of its high enzyme immobilization ability and stability. The method for immobilizing the enzyme is not particularly limited, and a known method can be used. For example, the methods such as the carrier binding method, the cross-linking method and the entrapping method described in “Immobilized Enzyme” (edited by Ichiro Chibata, Kodansha Scientific, 1975) can be mentioned.

A method for producing an immobilized enzyme membrane will be described by taking an example of immobilizing a monoamine oxidase on acetyl cellulose. Triacetyl cellulose is dissolved in dichloromethane, glutaraldehyde is added, and then 1,8-diamino-4-aminomethyloctane is added, and the mixture is thinly cast into a film on a glass plate or the like and dried. The membrane is reacted with glutaraldehyde and then contacted with a solution of monoamine oxidase to immobilize the monoamine oxidase.

The pH at the time of contacting the monoamine oxidase with the acetylcellulose membrane is generally in the range of pH 5 to 9,
Particularly, a pH range of 6.5 to 7.5 is suitable. As the buffer solution for maintaining the pH, a phosphate buffer solution, a GOOD buffer solution or the like can be preferably used. The temperature at that time is not particularly limited as long as the enzyme is stable, but specifically, it is in the range of 1 to 15 ° C, particularly 2 to 5 ° C.
The range of 0 ° C is suitable, and the reaction time is preferably in the range of 30 minutes to 3 hours, particularly preferably in the range of 1 to 2 hours.

The electrode used in the present invention is not particularly limited as long as it can detect an increase / decrease of a substance during the reaction catalyzed by the above-mentioned monoamine oxidase and convert it into an electric signal, and a known electrode is used. it can. For example, "immobilized enzyme"
(Edited by Ichiro Chibata, Kodansha Scientific, 1975
1), "Biosensor" (edited by Shuichi Suzuki, Kodansha Scientific, 1984), oxygen electrodes, hydrogen peroxide electrodes, ammonia electrodes and the like. The above-mentioned immobilized enzyme membrane is attached to such an electrode to prepare an enzyme electrode.

The amine compound used in the present invention is not particularly limited as long as it can serve as a substrate for the oxidative deamination reaction by a monoamine oxidase. Specific examples of the amine compound include benzylamine, p-fluorobenzylamine, phenethylamine, 1-aminobutane, 1
-Aminopentane, 1-aminohexane and the like,
Particularly, benzylamine is preferable in terms of affinity with the enzyme.

The amine compound may be present when the immobilized enzyme membrane comes into contact with the subject, and the method is not limited. It may be added to the buffer solution in advance, or an amine compound aqueous solution may be separately added to the enzyme reaction cell.

In the present invention, the reactant is a substance consumed in the oxidation reaction of an amine compound by a monoamine oxidase according to the above reaction formula, and means an amine compound or oxygen. The reaction product is a substance produced by a monoamine oxidase, such as hydrogen peroxide, ammonia,
Alternatively, it refers to an aldehyde compound derived from the above amine compound. Oxygen, hydrogen peroxide, or ammonia is preferably measured as a reaction product or a reaction product because it can be easily measured. In particular, it is more preferable to measure oxygen or hydrogen peroxide because it is unlikely to be affected by coexisting substances.

The subject in the present invention is not particularly limited as long as it contains chlorine ions. For example, blood,
Examples thereof include serum, plasma, urine, cultures, culture fluids, intracellular fluids, foods containing miso soy sauce, and extracts thereof.

The amount of liquid in the reaction system when the analyte and the amine compound are brought into contact with the enzyme electrode in the present invention and reacted is
Although not particularly limited, it is usually in the range of 0.1 ml to 5 ml. The amine compound is used at a concentration of 0.01-100 mM,
It is preferably used at a concentration in the range of 0.5 to 10 mM. P of reaction system
H is not particularly limited as long as it has a pH at which the activity of monoamine oxidase is maintained at a high level, but generally, a pH range of 5 to 9 is preferable, and a pH range of 6 to 8 is particularly preferable. The buffer solution for maintaining the pH is not particularly limited as long as it does not contain chloride ions, but for example, phosphate buffer solution, GOOD buffer solution and the like can be preferably used. The reaction temperature is preferably in the range of 10 to 45 ° C, particularly 25 ° C to 37 ° C. Reaction time is 30 seconds ~
A range of 15 minutes is preferred, especially a range of 1 to 5 minutes.

In the method for quantifying chloride ion of the present invention,
When measuring oxygen as a reaction product in the oxidation reaction of an amine compound by a monoamine oxidase, for example, FIG.
It is possible to use an enzyme electrode in which an oxygen electrode and an immobilized enzyme membrane are combined as shown in FIG. The immobilized enzyme membrane is immobilized on the surface of the oxygen electrode using an O-ring or the like. Working electrode,
Known substances can be used for the counter electrode, the oxygen permeable membrane, and the electrolyte solution. For example, a platinum electrode can be preferably used as the working electrode, a silver electrode as the counter electrode, a polytetrafluoroethylene film or the like as the oxygen permeable membrane, and a phosphate buffer solution or the like as the electrolyte solution. When a reaction solution consisting of the analyte, amine compound, and buffer solution is brought into contact with such an enzyme electrode, oxygen in the reaction solution permeates the immobilized enzyme membrane and oxygen permeable membrane and is reduced on the working electrode, resulting in oxygen concentration. Produces a reduction current proportional to. By measuring this reduction current value, the rate of decrease (amount) of oxygen due to the enzymatic reaction is quantified.

When hydrogen peroxide, which is a product of the oxidation reaction of an amine compound by a monoamine oxidase, is measured, for example, a hydrogen peroxide electrode and an immobilized enzyme membrane as shown in FIG. 2 are combined. Enzyme electrodes can be used. The immobilized enzyme membrane is immobilized on the surface of the hydrogen peroxide electrode using an O-ring or the like. Known substances can be used for the working electrode, the counter electrode, the porous polymer membrane, and the electrolyte solution. For example, the working electrode is a platinum electrode, the counter electrode is a silver electrode, the porous polymer membrane is a cellulose membrane, a polyethylene membrane or the like, and the electrolyte solution is a phosphate buffer or the like.
Each can be preferably used. When a reaction solution consisting of the analyte, amine compound, and buffer solution is brought into contact with such an enzyme electrode, hydrogen peroxide generated in the immobilized enzyme membrane permeates the porous polymer membrane and is oxidized on the working electrode. And produces an oxidation current proportional to the hydrogen peroxide concentration. By measuring this oxidation current value, the rate of increase (amount) of hydrogen peroxide due to the enzymatic reaction
Is quantified.

When ammonia, which is a product of the oxidation reaction of an amine compound by a monoamine oxidase, is measured, for example, an enzyme electrode having a combination of an ammonia electrode and an immobilized enzyme membrane as shown in FIG. 3 is used. can do. The immobilized enzyme membrane is immobilized on the surface of the ammonia electrode using an O-ring or the like. pH measuring glass electrode, reference electrode,
Known substances can be used for the hydrophobic porous membrane and the internal liquid. For example, a commercially available pH measuring glass electrode, a silver / silver chloride electrode as a reference electrode, polyvinylidene fluoride as a hydrophobic porous film, and an ammonium chloride solution as an internal liquid can be preferably used. . When a reaction solution consisting of the analyte, the amine compound, and the buffer solution is brought into contact with such an enzyme electrode, the ammonia produced in the immobilized enzyme membrane is
Permeation through the hydrophobic porous membrane changes the pH of the internal liquid,
The glass electrode for pH measurement responds to the change, resulting in a potential difference.
By measuring this potential difference value, the rate of increase (amount) of ammonia due to the enzymatic reaction is quantified.

Typical quantification of chloride ion concentration by the method of quantifying chloride ion of the present invention will be described with reference to FIG. The buffer solution is sent to the enzyme reaction cell by a constant flow pump. To the enzyme reaction cell filled with the buffer solution, a certain amount of the analyte is added from the sample injection tube, and then a certain amount of the amine compound is added from the amine compound injection tube. The enzyme reaction cell is equipped with a stirrer and a stirring motor to mix the analyte, the amine compound and the buffer solution. An enzyme electrode equipped with an immobilized monoamine oxidase membrane is attached to the enzyme reaction cell, and its output is recorded by a recorder via a potentiostat when measuring oxygen or hydrogen peroxide. When measuring ammonia, it is recorded on a recorder via a potentiometer. From the output of the enzyme electrode, the amount of decrease in oxygen, usually the rate of decrease, or the amount of increase in hydrogen peroxide or ammonia, usually the rate of increase, is quantified. The buffer solution, the enzyme reaction cell, and the enzyme electrode are kept at a constant temperature in a constant temperature bath. The liquid after the measurement is discharged through the waste liquid line.

The quantification of chlorine ion is typically carried out by using a standard solution of known chloride ion concentration prepared using a sodium chloride solution, a potassium chloride solution or the like prior to the measurement of an analyte of the present invention. According to the quantification method, for example, a calibration curve of oxygen decrease rate (amount) or hydrogen peroxide or ammonia increase rate (amount) and chloride ion concentration is created, and this test curve is used to measure the chloride ion of the subject. Calculate the concentration. In the measurement of the rate of increase in oxygen (amount) or the rate of increase in hydrogen peroxide (amount), the current value proportional to the amount of oxygen or the amount of hydrogen peroxide is directly measured as the respective rate of increase (amount). Since this can be obtained, it is common to prepare a calibration curve using this current value and use it for quantification. Similarly, in measuring the rate of increase (amount) of ammonia, a calibration curve is prepared using the potential difference value proportional to the amount of ammonia and used for quantification.

The method for quantifying chloride ion of the present invention comprises immobilizing an Aspergillus niger-derived monoamine oxidase on an acetyl cellulose membrane in terms of stability, accuracy, quantification and operability of an enzyme electrode. Using an enzyme electrode that is a combination of the immobilized enzyme membrane and an oxygen electrode or hydrogen peroxide electrode, the analyte is brought into contact with the immobilized enzyme membrane in the presence of benzylamine, and the oxidation reaction of benzylamine by monoamine oxidase is accompanied. It is preferably carried out by measuring the output of the enzyme electrode corresponding to the decrease in oxygen or the increase in hydrogen peroxide.

[0026]

[Function] Monoamine oxidase catalyzes the formation reaction of aldehyde compounds, ammonia, and hydrogen peroxide by oxidative deamination using an amine compound as a substrate, but when chlorine ion is present in the reaction solution, it depends on the concentration. Then the activity changes. By measuring oxygen as a reaction product or hydrogen peroxide, ammonia as a reaction product, the concentration of chlorine ions in the analyte introduced into the reaction solution can be quantified.

[0027]

INDUSTRIAL APPLICABILITY Since the method for quantifying chloride ion of the present invention uses an immobilized enzyme, the enzyme can be used repeatedly. Moreover, since an enzyme electrode composed of a combination of an immobilized enzyme and an electrode is used, it has become possible to accurately quantify chloride ion by a simple operation.

[0028]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Reference Example Preparation of monoamine oxidase 3.0% glucose, 0.3% sodium nitrate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate, 0.1% yeast extract, 0.02
Prepare 10 Sakaguchi flasks containing 100 ml of pre-culture medium (pH 5.0) consisting of an antifoaming agent, and use Aspergillus niger (As
pergillus niger, ATCC28325) was inoculated and cultured with shaking at 30 ° C. and 110 rpm for stirring overnight. The culture broth for 10 Sakaguchi flasks was transferred to a jar fermenter charged with 20 liters of the preculture broth, and cultured overnight at 30 ° C., agitation speed 200 rpm, and aeration 25 liter / min, and then the cells were collected. Then, inoculate into a jar fermenter charged with 160 l of preculture liquid, 30 ℃, stirring rotation speed 200 rpm, aeration 180 l / m
After culturing overnight in in vitro, the cells were harvested, 3.0% glucose, 0.1%
160 l of main culture medium (pH 5.0) consisting of butylamine, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate, 0.02% defoamer
Bacteria were inoculated into the jar fermenter that had been prepared, and at 30 ° C,
The cells were collected by performing main culture overnight at a stirring rotation speed of 200 rpm and aeration of 180 l / min.

About 5 kg (wet cell weight) of the obtained cells is 50
The cells were suspended in 20 mM phosphate buffer (pH 7.5) (1), and the cells were disrupted using a Dynomill cell disruptor. An insoluble matter was removed from the disrupted liquid by a continuous centrifuge to obtain a supernatant liquid.

20 mM phosphate buffer (pH
3 l of DEAE-cellulose (manufactured by Whatman) that had been equilibrated in 7.0) was added, and the mixture was left overnight at 4 ° C with gentle stirring. This solution was subjected to suction filtration to obtain a protein-adsorbed resin, which was thoroughly washed and then packed in a column. 0.
The adsorbed protein was eluted with 20 mM phosphate buffer containing 2 M ammonium sulfate, the active fraction was recovered, and desalted by gel filtration column chromatography.

Next, the desalted active fraction was passed through a 1 liter DEAE-cellulose column equilibrated with 20 mM phosphate buffer to adsorb proteins, and after washing the column with the same buffer, the ammonium sulfate concentration was adjusted. The protein was eluted with a linear increase from 0 M to 0.2 M and the active fraction was collected.

The active fraction was concentrated by ultrafiltration and equilibrated with 10 mM phosphate buffer containing 0.1 M ammonium sulfate.
It was passed through a 3 l Sephacryl S-400 (manufactured by Pharmacia) column to collect the active fraction, which was used as a final purified sample.

From about 5 kg (wet cell weight) of bacterial cells, 900 units (specific activity = 1.5 units / mg protein) of monoamine oxidase were obtained.

Example 1 Production of Immobilized Enzyme Membrane 250 mg of triacetyl cellulose was added to 5 ml of dichloromethane and dissolved by stirring for about 15 minutes. To this solution, with stirring, 0.05 ml of 50% glutaraldehyde solution was added. After stirring this solution for 15 minutes, 1 ml of 1,8-diamino-4-aminomethyloctane was added, mixed well and spread thinly on a glass plate. After air-drying at room temperature for 2 days, soak in deionized water to peel off the film,
Washed twice. This membrane was immersed in 100 ml of a 0.1 M phosphate buffer containing 1% glutaraldehyde, pH 6.8, treated at room temperature for 1 hour, and then washed 5 times with deionized water.

5 ml of monoamine oxidase (1 unit / m
The membrane treated with glutaraldehyde was immersed in 0.1 M phosphate buffer (pH 6.8) containing l) and reacted at 4 ° C for 1 hour. 0.
The membrane was thoroughly washed with 1M phosphate buffer, pH 6.8 to obtain an immobilized monoamine oxidase membrane.

Example 2 The immobilized enzyme membrane prepared in Example 1 was attached to an oxygen electrode,
An enzyme electrode having the structure shown in FIG. 1 was produced. The device shown in FIG. 4 was produced by incorporating this enzyme electrode. The buffer, enzyme reaction cell, and enzyme electrode were kept at 37 ° C. As a sample, 0
, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 3
00, 400 and 500 mM sodium chloride solutions were prepared and measured. 1.9 ml of 0.1 M phosphate buffer, pH 6.8 was injected into the enzyme reaction cell, and 0.05 ml of sample was injected into the enzyme reaction cell. Next, 0.1 ml of 0.1 M benzylamine aqueous solution (pH adjusted to 6.8 with phosphoric acid) was added to the enzyme reaction cell, the output (current) of the enzyme electrode was measured for 3 minutes, and the amount of change in current value per minute was measured. (ΔμA) was calculated. The horizontal axis shows the chloride ion concentration, and the vertical axis shows the reciprocal of ΔμA (1 / ΔμA). The result is shown in FIG. As is clear from FIG. 5, a good linear relationship was obtained in the chloride ion concentration range of 0 to 500 mM.

Example 3 The method described in Example 2 except that the immobilized enzyme membrane prepared in Example 1 was attached to a hydrogen peroxide electrode and the enzyme electrode having the structure shown in FIG. 2 was used. Measured by 1 /
ΔμA was calculated. Chlorine ion concentration on the horizontal axis, Δμ on the vertical axis
The reciprocal of A (1 / ΔμA) was taken and plotted. The results are shown in FIG. As is clear from FIG. 6, a good linear relationship was obtained in the chloride ion concentration range of 0 to 500 mM.

[Brief description of drawings]

FIG. 1 is a schematic cross-sectional configuration diagram of an enzyme electrode used when measuring oxygen concentration in the method for quantifying chlorine ions of the present invention.

FIG. 2 is a schematic cross-sectional configuration diagram of an enzyme electrode used for measuring the concentration of hydrogen peroxide by the method for quantifying chlorine ions of the present invention.

FIG. 3 is a schematic cross-sectional configuration diagram of an enzyme electrode used when measuring the concentration of ammonia in the method for quantifying chlorine ions of the present invention.

FIG. 4 is a schematic view of an apparatus for carrying out the method for quantifying chloride ion of the present invention.

FIG. 5 is a diagram showing the relationship between chlorine ions and the reciprocal of the change in current value per minute (1 / ΔμA) when the oxygen concentration is measured by the method for quantifying chlorine ions of the present invention.

FIG. 6 is a diagram showing the relationship between chlorine ions and the reciprocal of the change in current value per minute (1 / ΔμA) when the hydrogen peroxide concentration was measured by the method for quantifying chlorine ions of the present invention. is there.

[Explanation of symbols]

 1 Immobilized enzyme membrane 2 Oxygen permeable membrane 3 Working electrode 4 Counter electrode 5 Electrolyte solution 6 Power supply (0.5-0.8V) 7 Potentiostat 8 Immobilized enzyme membrane 9 Porous polymer membrane 10 Working electrode 11 Counter electrode 12 Electrolyte solution 13 Power supply ( 0.3-0.5V) 14 Potentiostat 15 Immobilized enzyme membrane 16 Hydrophobic porous membrane 17 pH measuring glass electrode 18 Reference electrode 19 Internal solution 20 Potentiometer 21 Buffer solution 22 Constant flow pump 23 Enzyme reaction cell 24 Sample injection tube 25 Amine compound injection pipe 26 Stirrer 27 Stirring motor 28 Enzyme electrode with immobilized enzyme membrane 29 Potentiostat or potentiometer 30 Recorder 31 Constant temperature bath 32 Waste liquid line

Claims (1)

[Claims] 1. An enzyme electrode comprising a combination of an immobilized enzyme membrane in which a monoamine oxidase is immobilized on a membrane polymer compound and an electrode is used, and an analyte containing chlorine ions is immobilized on the immobilized enzyme membrane of the enzyme electrode. Chloride ion content was measured by contacting in the presence of an amine compound and measuring the output of the enzyme electrode generated in response to the decrease amount of the reaction product or the increase amount of the reaction product accompanying the oxidation reaction of the amine compound by the monoamine oxidase. A method for quantifying chlorine ion, which comprises determining.